- Email: [email protected]
Modification of calf thymus DNA by methyl methanesulfonate. Quantitative determination of 7-methyldeoxyguanosine by mass spectrometry
295
Chem.-Biol. Interactions, 57 (1986) 295--300 Elsevier Scientific Publishers Ireland Ltd.
MODIFICATION OF CALF THYMUS DNA BY METHYL METHANESULFONATE. QUANTITATIVE DETERMINATION OF 7-METHYLDEOXYGUANOSINE BY MASS SPECTROMETRY
CHING-JER CHANG a, DENNIS J. ASHWORTH a, XIANG-YU JIANG b and R. GRAHAM COOKS b
ILEANA
ISERN-FLECHA b,
aDepartment o f Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences and bDepartment of Chemistry, Purdue University, West Lafayette, IN 47907 (U.S.A.) (Received September 3rd, 1985) (Accepted November 22, 1985)
SUMMARY
Quantitation of 7-methyldeoxyguanosine (m7dG) produced in the in vitro methyl methanesulfonate (MeMS) action on calf-thymus DNA is achieved by enzymatic degradation, liquid chromatographic separation and chemical ionization mass spectrometry. The total degree of methylation, measured by uptake of [~4C]MeMS was 0.35%. Mass spectral analysis shows that mTdG constitutes 84% of the total methylated product. It is also shown that tandem mass spectrometry allows detection of mTdG, as the protonated base, down to 1 pmol level, suggesting that MS/MS analysis can be the method of choice in quantitation of the adducts of in vivo DNA modifications.
Key words: Calf thymus DNA -- 7-Methyldeoxyguanosine -- Mass spectrometry -- Tandem mass spectrometry INTRODUCTION
The alkylation of DNA is the primary basis for the biological activity of alkylating agents [1--3]. Different methods have been developed to determine the sites of DNA modification [1--3]. Carbon-13 nuclear magnetic resonance is effective in determining the sites of reaction of methylating agents with nucleic acids without prior degradation or separation processes [4--6]. This method requires a fairly large amount of material and a high Abbreviations: HPLC, high pressure liquid chromatography; m~dG, 7-methyldeoxyguanosine; MeMS, methyl methanesulfonate; MeMU, methylnitrosourea. 0009-2797/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
296 degree of reaction due to the inherent low sensitivity of ~3C-NMR. Recently, high pressure liquid chromatography (HPLC) has also been successfully developed to analyze methylated DNA products by us and others [7]. However, the liquid chromatogram itself provides little structural information. We have therefore developed a mass spectrometric method for qualitative and quantitative analysis of methylated DNA adducts [8,9]. Alkylation at the N-7 position of guanine by dimethyl sulfate is used in the selective cleavage of deoxyguanosine in DNA sequence analysis [10]. A great deal of interest has been directed to the determination of 7-alkyldeoxyguanine since its isolation from mustard-treated microorganisms [11]. Mass spectrometry is rapidly becoming more widely applied in DNA research and has been used in detection of this compound. Electron impact mass spectrometry was utilized in elucidating the mode of formation of 7-methylguanine in the dimethylnitrosamine-treated rats [ 12 ] and N-methylN'-nitro-N-nitrosoguanidine-treated Escherichia coli [13]. Field desorption [14], 2s2Cf desorption [15] and secondary-ion [8] mass spectra of 7methylguanosine and/or 7-methyldeoxyguanine have also being measured. The chemical ionization mass spectrometry and HPLC methodology used here has been previously applied in the quantitative analysis of modified nucleosides by methylnitrosourea (MENU) [16]. In the former experiments, 2.43% methyl incorporation occurred when purified calf thymus DNA was treated with MENU. The major product in in vitro alkylation with MeNU was mT
Calf thymus DNA (Type 1, Sigma Chemical Company, No. D-1501) was purified prior to alkylation by extensive dialysis against double distilled water and was shown to be 75--80% double-stranded. The purified material (2.79 × 10 -2 mmol, as determined by UV analysis, e260:11 653 and calculated from the known composition based on an average molecular weight of sodium salt of nucleoside monophosphate (349 Daltons)] was added to a pH stat reaction vessel and dissolved in 5.0 ml of double distilled water (pH 7.0). To this solution was then added 2.88 × 10 -5 mmol of ~2C/~4CMeMS (spec. act. 6.39 pCi/mmol) and it was ~llowed to react for 3 h on the pH-stat at room temperature. The pH of the solution was maintained at 7.0 by addition of 0.1 N NaOH. After 3 h the solution was transferred to a
297
dialysis membrane and dialyzed against 500 ml double distilled water (pH 7.0), every hour until the radioactivity of the outside solution was no greater than normal background. Carbon-14 analysis of the methylated D N A indicated on incorporation of 0.35%. The D N A solution (2.53 × 10 -2 retool) was lyophilized and then added into 0.5 rnl of 0.1 M Tris buffer (pH 7.3) in a 10 ml test tube. Sixty units of DNase I (Type Ill, 1500 units/rag, Sigma), 3.06 units of alkaline phosphatase (Type Ill, 25 units/rag, Sigma), 0.0303 units of phosphodiesterase (snake venom Type Ill, 0.2 units/rag, Sigma) and 2.42 X 10 -2 ~mol of [CD3-methyl]mTdG in 0.15 ml of buffer solution were then added to the D N A solution. Enzymatic degradation was allowed to continue for 18 h at 37°C in a shaker bath at which time the degraded sample was filtered through a 0.4 ~ m filter and lyophilized. The dry nucleosides were then dissolved in 300/~I of water and 100 ~l of solution injected onto the H P L C (3 times). The column used was a Waters radial-compression C1s cartridge. A solution 0.05 M in a m m o n i u m formate and 3 m M in tetraethylammoniurn chloride was used as mobile phase, with a gradient elution (Waters 660 programmer, program #7) of 0--15% methanol in 25 rain at a flow rate of 4 ml/min. Detection was accomplished by U V at 254 and 280 nm. W h e n the peak (R t = 8.9 rain, UV: A254/A280 = 0.72) corresponding to m T d G was identified, the eluting fraction was collected and lyophilized. Due to the large amount of buffer and ion-pair reagent present, the lyophilized powder was dissolved in 100. pl of water and applied to 1 g Ag-I-X8 cation exchange resin (H ÷ form) equilibrated with double distilled water. After application of the sample, the column was washed with 5 ml of water to remove formate and chloride ions and the nucleosides eluted with 1 % N H 4 O H . Fractions 22--25 [40 drops (1 ml)/fraction] were combined and lyophilized for mass spectral analysis. Mass spectral analysis was performed on a Finnigan 4000 mass spectrometer. Chemical ionization employed isobutane as reagent gas at a pressure of approx. 0.3 Tort. The dried m T d G fraction was dissolved in approx. 9 ~I H 2 0 in a rnicrocapillarytube and introduced via the direct insertion probe. Fast heating of the probe (50°--350°C in < 1 rain) optimized detection of the nucleosides. One scan was recorded every 2 s as the probe was heated. Twenty scans were then s u m m e d corresponding to 10 scans on each side of the m a x i m u m relativeion current. M S / M S (daughter) spectra of m T d G were obtained using a 4500 Finnigan triple quadrupole mass spectrometer. To lower the limits of detection further in M S / M S experiments, different experimental conditions were investigated for both the direct insertion probe and desorption chemical ionization probe. O p t i m u m experimental conditions for daughter ion spectra of the protonated bases are as follows: ammonia as reagent gas at a pressure of approx. 0.75 Tort, argon collision gas at a pressure of approx. 1.9 mTorr and a collision energy of approx. 16 eV. The direct insertion probe was heated to 400°C in less than a minute, while the desorption chemical ionization probe was heated to 700°C at a rate of 100°C/s. One scan was recorded
298 every 0.35 s as the probe was heated. In chemical ionization, 50 scans were summed corresponding to 25 scans on each side of the m a x i m u m ion current, while in desorption chemical ionization, only 6 scans were summed due to faster evaporation of the sample. RESULTS AND DISCUSSION
Reaction of calf t h y m u s DNA with MeMS for 3 h produced 0.35% m e t h y l incorporation as determined by 14C analysis. The deuterium-labeled mTdG was added as an internal reference for mass spectral analysis and as a marker for HPLC fractionation. This exogenous deuterated c o m p o u n d also served as a carrier, and so minimized the loss o f sample in transfer and fractionation processes. The primary objective of HPLC separation was not complete resolution of all nucleosides. The essential requirement of the system is the resolution of those structural isomers which can not be differentiated by mass spectrometry. Two other structural isomers [1-methyldeoxyguanosine (Rt = 18.6 rain, A:s4/A2,0 = 1.93) and O6-methyldeoxyguanosine (Rt = 27.3 rain, A254/A280 = 0.97)] of mTdG (Rt = 8.9 rain, A2s4/A2~0 = 0.72) were clearly resolved in the positive ion-pair revers~phase HPLC system. The chemical ionization mass spectrum of the HPLC fraction corresponding to m~dG is shown in Fig. 1. The product is detected as the protonated 7-methylguanine due to facile cleavage of the glycosyl bond. Quantitative determination of mTdG is readily made on the basis of the peak ratio (32.5% and 100% relative abundance, respectively) of the 7-CD3 guanine and 7-CH3 guanine and the k n o w n a m o u n t (24.2 nmol) of the
. o
100-
66
oH,
OH
8
50
mTdG 169
I
M/Z
I
130
!
!
150
r
I
170
Fig. 1. Mass spectral quantification of m~dG isolated from calf thymus DNA reacted with MeMS, IH~-methyl base (analyte) peak at m / z 166, 2Hs-methyl base (reference) peak at m / z 169.
299 166
10(
166
100
149
149 50
50' 124
I'r
N1/Z 125
124
145
165
r,4./z 12,5
145
165
Fig. 2. (a) Daughter spectrum of protonated 7-methylguanine from I 0 ng of m~dG (15 eV collision energy, 2.0 reTort argon collision gas). (b) Multiple reaction monitoring spectrum of protonated 7-methylguanine from 0.2 ng of mVdG (15 eV collision energy, 2.0 reTort argon collision gas).
deuterated reference compound. Knowing that 0.35% methyl incorporation occurs, the total number of moles of methylated nucleotide (assuming monomethylation only) is given as 0.35% of the total number of DNA moles (2.53 × 10-s), viz. 88.6 nmol. Of this amount, (24.2 X 100)/32.5 nmol, i.e. 74.5 nmol is mTdG. That is, the yield of mTdG with reference to all methylated products is 84%. These results compare favorably with 86% of mTdG (with reference to all methylated products) reported in in vitro methylation of calf-thymus DNA with MeMS, determined by radioactivity measurements [19l. The potential of tandem mass spectrometry in quantitative determinations of mTdG is evident from results obtained in optimizing its performance. Tandem mass spectrometry yields abundant fragment ions which are not characteristic of the mass spectrum. This zwitterionic nucleoside is convetted to the cationic form in the presence of p-toluenesulfonic acid (p-TSA) matrix which enhances the ion yield in chemical ionization and desorption chemical ionization (DCI) experiments by a factor of at least five times (from 0.0052 M p-TSA solution). However, chemical noise from the matrix limits its use in quantitating subnanogram levels of mTdG. As little as 1.0 ng (3 pmol) of mTdG has been detected from DCI daughter ion spectra of the protonated base (see Fig. 2a). The limits of detection are lowered further if multiple reaction monitoring [16] is used, and even 0.7 pmol of mTdG have been detected with this technique {see Fig. 2b). The selectivity and sensitivity provided by tandem mass spectrometry in detection of mTdG should make the technique suitable for future in vivo studies of chemically modified DNA. ACKNOWLEDGEMENTS
W e gratefully acknowledge financial support from the National Cancer Institute(CA 35904) and the Environmental Protection Agency (R 811138).
300 REFERENCES 1 P.L. Grover, Chemical Carcinogenesis and D N A , C R C Press, Boca Raton, Florida, 1979. 2 W. Lijinsky, Interaction with nucleic acids of carcinogenic and mutagenic N-nitroso compounds, Prog. Nucl. Acid. Res. Mol. Biol., 17 (1976) 247. 3 B. Singer and D. Grunberger, Molecular Biology of Mutagens and Carcinogens, Plenum Press, N e w York, 1983. 4 C.-J. Chang and C.-G. Lee, Direct study of alkylating agent-RNA interaction by 13C nuclear magnetic resonance spectroscopy, Cancer Res., 38 (1978) 3734. 5 C.-J. Chang and C.-G. Lee, Chemical modification of ribonucleic acid. A direct study by carbon-13 nuclear magnetic resonance spectroscopy, Biochemistry, 20 (1981) 2657. 6 C.-J. Chang, J. DaSilva Gomes and S.R. Byrn, Chemical modification of deoxyribonucleic acids: a direct study by carbon-13 nuclear magnetic resonance spectroscopy, J. Org. Chem., 48 (1983) 5151. 7 J. DaSilva Gomes and C.-J. Chang, Reverse-phase high performance liquid chromatography of chemically modified DNA, Anal. Biochem., 129 (1983) 387, and other references therein. 8 S.E. Unger, A.E. Schoen, R.G. Cooks, D.J. Ashworth, J. Dasilva Gomes and C.-J. Chang, Identification of modified nucleosides by secondary-ion mass spectrometry, J. Org. Chem., 46 (1981) 4765. 9 D.J. Ashworth, C.-J. Chang, S.E. Unger and R.G. Cooks, Chemical modification of polynucleotides. Quantitative studies o f polycytidylic acid by nuclear magnetic resonance spectroscopy and secondary-ion mass spectrometry, J. Org. Chem., 46 (1981) 4770. 10 A.M. Maxam and W. Gilbert, Sequencing end-labeled DNA with base-specific chemical cleavage, Methods Enzymol., 65 (1980) 499 11 P.D. Lawley, Effects of some chemical mutagens and carcinogens on nucleic acids, Prog. Nucl. Acid Mol. Biol., 5 (1966) 89. 12 W. Lijinsky, J. Loo, and A.E. Ross, Mechanism of alkylation of nucleic acids b y nitrosodimethylamine, Nature, 218 (1968) 1174. 13 R.F. Gomez, M. Johnston and A.J. Sinskey, The mode of action of N-methyl-N *nitro-N-nitroguanidine VII. The transfer of the methyl group of N-methyl-N'-nitroN-nitrosoguanidine, Biochim. Biophys. Acta, 269 (1972) 276. 14 C. Fenselau, M.-N. Kan, G,H. Sack Jr., G.W. Wood, D.E. Schmidt Jr. and P.-Y. Lau, Field desorption mass spectrometry as a complementary technique for characterization of alkylated nucleosides, Adv. Mass Spectrom., 7 (1978) 1572. 15 Y. Le Beyec, S. Della Negra, C. Deprum, P. Vigny and y.M. Ginot, Mass determination of molecules of biological interest b y fast heavy ions induced desorption mass spectrometry, Rev, Phys. Appl., 151 (1980) 1631. 16 D.J. Ashworth, W.M. Baird and C.-J. Chang, J.D. Ciupek, K.L. Busch and R.G. Cooks, Chemical modification of nucleic acids. Methylation of calf thymus DNA investigated by mass spectrometry and liquid chromatography, Biomed. Mass. Spectrom., (1986) in press. 17 M.A. Baldwin and F.W. McLafferty, Direct chemical ionization of relativelyinovolatile samples. Application to underivatized oligopeptides, Org. Mass Spectrom., 7 (1973) 1353. 18 E.L. Esmans, E.J. Freyne, J.H. Vanbroeckhoven and F.C. Alderweireldt, Chemlcal ionization desorption mass spectrometry as an additional tool for the structure elucidation of nucleosides, Biomed. Mass Spectrom., 7 (1980) 377. 19 P.D. Lawley and P. Brookes, Further studies on the alkylation of nucleic acids and their constituent nucleotides, Biochem. J., 89 (1963) 127.
Chem.-Biol. Interactions, 57 (1986) 295--300 Elsevier Scientific Publishers Ireland Ltd.
MODIFICATION OF CALF THYMUS DNA BY METHYL METHANESULFONATE. QUANTITATIVE DETERMINATION OF 7-METHYLDEOXYGUANOSINE BY MASS SPECTROMETRY
CHING-JER CHANG a, DENNIS J. ASHWORTH a, XIANG-YU JIANG b and R. GRAHAM COOKS b
ILEANA
ISERN-FLECHA b,
aDepartment o f Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences and bDepartment of Chemistry, Purdue University, West Lafayette, IN 47907 (U.S.A.) (Received September 3rd, 1985) (Accepted November 22, 1985)
SUMMARY
Quantitation of 7-methyldeoxyguanosine (m7dG) produced in the in vitro methyl methanesulfonate (MeMS) action on calf-thymus DNA is achieved by enzymatic degradation, liquid chromatographic separation and chemical ionization mass spectrometry. The total degree of methylation, measured by uptake of [~4C]MeMS was 0.35%. Mass spectral analysis shows that mTdG constitutes 84% of the total methylated product. It is also shown that tandem mass spectrometry allows detection of mTdG, as the protonated base, down to 1 pmol level, suggesting that MS/MS analysis can be the method of choice in quantitation of the adducts of in vivo DNA modifications.
Key words: Calf thymus DNA -- 7-Methyldeoxyguanosine -- Mass spectrometry -- Tandem mass spectrometry INTRODUCTION
The alkylation of DNA is the primary basis for the biological activity of alkylating agents [1--3]. Different methods have been developed to determine the sites of DNA modification [1--3]. Carbon-13 nuclear magnetic resonance is effective in determining the sites of reaction of methylating agents with nucleic acids without prior degradation or separation processes [4--6]. This method requires a fairly large amount of material and a high Abbreviations: HPLC, high pressure liquid chromatography; m~dG, 7-methyldeoxyguanosine; MeMS, methyl methanesulfonate; MeMU, methylnitrosourea. 0009-2797/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
296 degree of reaction due to the inherent low sensitivity of ~3C-NMR. Recently, high pressure liquid chromatography (HPLC) has also been successfully developed to analyze methylated DNA products by us and others [7]. However, the liquid chromatogram itself provides little structural information. We have therefore developed a mass spectrometric method for qualitative and quantitative analysis of methylated DNA adducts [8,9]. Alkylation at the N-7 position of guanine by dimethyl sulfate is used in the selective cleavage of deoxyguanosine in DNA sequence analysis [10]. A great deal of interest has been directed to the determination of 7-alkyldeoxyguanine since its isolation from mustard-treated microorganisms [11]. Mass spectrometry is rapidly becoming more widely applied in DNA research and has been used in detection of this compound. Electron impact mass spectrometry was utilized in elucidating the mode of formation of 7-methylguanine in the dimethylnitrosamine-treated rats [ 12 ] and N-methylN'-nitro-N-nitrosoguanidine-treated Escherichia coli [13]. Field desorption [14], 2s2Cf desorption [15] and secondary-ion [8] mass spectra of 7methylguanosine and/or 7-methyldeoxyguanine have also being measured. The chemical ionization mass spectrometry and HPLC methodology used here has been previously applied in the quantitative analysis of modified nucleosides by methylnitrosourea (MENU) [16]. In the former experiments, 2.43% methyl incorporation occurred when purified calf thymus DNA was treated with MENU. The major product in in vitro alkylation with MeNU was mT
Calf thymus DNA (Type 1, Sigma Chemical Company, No. D-1501) was purified prior to alkylation by extensive dialysis against double distilled water and was shown to be 75--80% double-stranded. The purified material (2.79 × 10 -2 mmol, as determined by UV analysis, e260:11 653 and calculated from the known composition based on an average molecular weight of sodium salt of nucleoside monophosphate (349 Daltons)] was added to a pH stat reaction vessel and dissolved in 5.0 ml of double distilled water (pH 7.0). To this solution was then added 2.88 × 10 -5 mmol of ~2C/~4CMeMS (spec. act. 6.39 pCi/mmol) and it was ~llowed to react for 3 h on the pH-stat at room temperature. The pH of the solution was maintained at 7.0 by addition of 0.1 N NaOH. After 3 h the solution was transferred to a
297
dialysis membrane and dialyzed against 500 ml double distilled water (pH 7.0), every hour until the radioactivity of the outside solution was no greater than normal background. Carbon-14 analysis of the methylated D N A indicated on incorporation of 0.35%. The D N A solution (2.53 × 10 -2 retool) was lyophilized and then added into 0.5 rnl of 0.1 M Tris buffer (pH 7.3) in a 10 ml test tube. Sixty units of DNase I (Type Ill, 1500 units/rag, Sigma), 3.06 units of alkaline phosphatase (Type Ill, 25 units/rag, Sigma), 0.0303 units of phosphodiesterase (snake venom Type Ill, 0.2 units/rag, Sigma) and 2.42 X 10 -2 ~mol of [CD3-methyl]mTdG in 0.15 ml of buffer solution were then added to the D N A solution. Enzymatic degradation was allowed to continue for 18 h at 37°C in a shaker bath at which time the degraded sample was filtered through a 0.4 ~ m filter and lyophilized. The dry nucleosides were then dissolved in 300/~I of water and 100 ~l of solution injected onto the H P L C (3 times). The column used was a Waters radial-compression C1s cartridge. A solution 0.05 M in a m m o n i u m formate and 3 m M in tetraethylammoniurn chloride was used as mobile phase, with a gradient elution (Waters 660 programmer, program #7) of 0--15% methanol in 25 rain at a flow rate of 4 ml/min. Detection was accomplished by U V at 254 and 280 nm. W h e n the peak (R t = 8.9 rain, UV: A254/A280 = 0.72) corresponding to m T d G was identified, the eluting fraction was collected and lyophilized. Due to the large amount of buffer and ion-pair reagent present, the lyophilized powder was dissolved in 100. pl of water and applied to 1 g Ag-I-X8 cation exchange resin (H ÷ form) equilibrated with double distilled water. After application of the sample, the column was washed with 5 ml of water to remove formate and chloride ions and the nucleosides eluted with 1 % N H 4 O H . Fractions 22--25 [40 drops (1 ml)/fraction] were combined and lyophilized for mass spectral analysis. Mass spectral analysis was performed on a Finnigan 4000 mass spectrometer. Chemical ionization employed isobutane as reagent gas at a pressure of approx. 0.3 Tort. The dried m T d G fraction was dissolved in approx. 9 ~I H 2 0 in a rnicrocapillarytube and introduced via the direct insertion probe. Fast heating of the probe (50°--350°C in < 1 rain) optimized detection of the nucleosides. One scan was recorded every 2 s as the probe was heated. Twenty scans were then s u m m e d corresponding to 10 scans on each side of the m a x i m u m relativeion current. M S / M S (daughter) spectra of m T d G were obtained using a 4500 Finnigan triple quadrupole mass spectrometer. To lower the limits of detection further in M S / M S experiments, different experimental conditions were investigated for both the direct insertion probe and desorption chemical ionization probe. O p t i m u m experimental conditions for daughter ion spectra of the protonated bases are as follows: ammonia as reagent gas at a pressure of approx. 0.75 Tort, argon collision gas at a pressure of approx. 1.9 mTorr and a collision energy of approx. 16 eV. The direct insertion probe was heated to 400°C in less than a minute, while the desorption chemical ionization probe was heated to 700°C at a rate of 100°C/s. One scan was recorded
298 every 0.35 s as the probe was heated. In chemical ionization, 50 scans were summed corresponding to 25 scans on each side of the m a x i m u m ion current, while in desorption chemical ionization, only 6 scans were summed due to faster evaporation of the sample. RESULTS AND DISCUSSION
Reaction of calf t h y m u s DNA with MeMS for 3 h produced 0.35% m e t h y l incorporation as determined by 14C analysis. The deuterium-labeled mTdG was added as an internal reference for mass spectral analysis and as a marker for HPLC fractionation. This exogenous deuterated c o m p o u n d also served as a carrier, and so minimized the loss o f sample in transfer and fractionation processes. The primary objective of HPLC separation was not complete resolution of all nucleosides. The essential requirement of the system is the resolution of those structural isomers which can not be differentiated by mass spectrometry. Two other structural isomers [1-methyldeoxyguanosine (Rt = 18.6 rain, A:s4/A2,0 = 1.93) and O6-methyldeoxyguanosine (Rt = 27.3 rain, A254/A280 = 0.97)] of mTdG (Rt = 8.9 rain, A2s4/A2~0 = 0.72) were clearly resolved in the positive ion-pair revers~phase HPLC system. The chemical ionization mass spectrum of the HPLC fraction corresponding to m~dG is shown in Fig. 1. The product is detected as the protonated 7-methylguanine due to facile cleavage of the glycosyl bond. Quantitative determination of mTdG is readily made on the basis of the peak ratio (32.5% and 100% relative abundance, respectively) of the 7-CD3 guanine and 7-CH3 guanine and the k n o w n a m o u n t (24.2 nmol) of the
. o
100-
66
oH,
OH
8
50
mTdG 169
I
M/Z
I
130
!
!
150
r
I
170
Fig. 1. Mass spectral quantification of m~dG isolated from calf thymus DNA reacted with MeMS, IH~-methyl base (analyte) peak at m / z 166, 2Hs-methyl base (reference) peak at m / z 169.
299 166
10(
166
100
149
149 50
50' 124
I'r
N1/Z 125
124
145
165
r,4./z 12,5
145
165
Fig. 2. (a) Daughter spectrum of protonated 7-methylguanine from I 0 ng of m~dG (15 eV collision energy, 2.0 reTort argon collision gas). (b) Multiple reaction monitoring spectrum of protonated 7-methylguanine from 0.2 ng of mVdG (15 eV collision energy, 2.0 reTort argon collision gas).
deuterated reference compound. Knowing that 0.35% methyl incorporation occurs, the total number of moles of methylated nucleotide (assuming monomethylation only) is given as 0.35% of the total number of DNA moles (2.53 × 10-s), viz. 88.6 nmol. Of this amount, (24.2 X 100)/32.5 nmol, i.e. 74.5 nmol is mTdG. That is, the yield of mTdG with reference to all methylated products is 84%. These results compare favorably with 86% of mTdG (with reference to all methylated products) reported in in vitro methylation of calf-thymus DNA with MeMS, determined by radioactivity measurements [19l. The potential of tandem mass spectrometry in quantitative determinations of mTdG is evident from results obtained in optimizing its performance. Tandem mass spectrometry yields abundant fragment ions which are not characteristic of the mass spectrum. This zwitterionic nucleoside is convetted to the cationic form in the presence of p-toluenesulfonic acid (p-TSA) matrix which enhances the ion yield in chemical ionization and desorption chemical ionization (DCI) experiments by a factor of at least five times (from 0.0052 M p-TSA solution). However, chemical noise from the matrix limits its use in quantitating subnanogram levels of mTdG. As little as 1.0 ng (3 pmol) of mTdG has been detected from DCI daughter ion spectra of the protonated base (see Fig. 2a). The limits of detection are lowered further if multiple reaction monitoring [16] is used, and even 0.7 pmol of mTdG have been detected with this technique {see Fig. 2b). The selectivity and sensitivity provided by tandem mass spectrometry in detection of mTdG should make the technique suitable for future in vivo studies of chemically modified DNA. ACKNOWLEDGEMENTS
W e gratefully acknowledge financial support from the National Cancer Institute(CA 35904) and the Environmental Protection Agency (R 811138).
300 REFERENCES 1 P.L. Grover, Chemical Carcinogenesis and D N A , C R C Press, Boca Raton, Florida, 1979. 2 W. Lijinsky, Interaction with nucleic acids of carcinogenic and mutagenic N-nitroso compounds, Prog. Nucl. Acid. Res. Mol. Biol., 17 (1976) 247. 3 B. Singer and D. Grunberger, Molecular Biology of Mutagens and Carcinogens, Plenum Press, N e w York, 1983. 4 C.-J. Chang and C.-G. Lee, Direct study of alkylating agent-RNA interaction by 13C nuclear magnetic resonance spectroscopy, Cancer Res., 38 (1978) 3734. 5 C.-J. Chang and C.-G. Lee, Chemical modification of ribonucleic acid. A direct study by carbon-13 nuclear magnetic resonance spectroscopy, Biochemistry, 20 (1981) 2657. 6 C.-J. Chang, J. DaSilva Gomes and S.R. Byrn, Chemical modification of deoxyribonucleic acids: a direct study by carbon-13 nuclear magnetic resonance spectroscopy, J. Org. Chem., 48 (1983) 5151. 7 J. DaSilva Gomes and C.-J. Chang, Reverse-phase high performance liquid chromatography of chemically modified DNA, Anal. Biochem., 129 (1983) 387, and other references therein. 8 S.E. Unger, A.E. Schoen, R.G. Cooks, D.J. Ashworth, J. Dasilva Gomes and C.-J. Chang, Identification of modified nucleosides by secondary-ion mass spectrometry, J. Org. Chem., 46 (1981) 4765. 9 D.J. Ashworth, C.-J. Chang, S.E. Unger and R.G. Cooks, Chemical modification of polynucleotides. Quantitative studies o f polycytidylic acid by nuclear magnetic resonance spectroscopy and secondary-ion mass spectrometry, J. Org. Chem., 46 (1981) 4770. 10 A.M. Maxam and W. Gilbert, Sequencing end-labeled DNA with base-specific chemical cleavage, Methods Enzymol., 65 (1980) 499 11 P.D. Lawley, Effects of some chemical mutagens and carcinogens on nucleic acids, Prog. Nucl. Acid Mol. Biol., 5 (1966) 89. 12 W. Lijinsky, J. Loo, and A.E. Ross, Mechanism of alkylation of nucleic acids b y nitrosodimethylamine, Nature, 218 (1968) 1174. 13 R.F. Gomez, M. Johnston and A.J. Sinskey, The mode of action of N-methyl-N *nitro-N-nitroguanidine VII. The transfer of the methyl group of N-methyl-N'-nitroN-nitrosoguanidine, Biochim. Biophys. Acta, 269 (1972) 276. 14 C. Fenselau, M.-N. Kan, G,H. Sack Jr., G.W. Wood, D.E. Schmidt Jr. and P.-Y. Lau, Field desorption mass spectrometry as a complementary technique for characterization of alkylated nucleosides, Adv. Mass Spectrom., 7 (1978) 1572. 15 Y. Le Beyec, S. Della Negra, C. Deprum, P. Vigny and y.M. Ginot, Mass determination of molecules of biological interest b y fast heavy ions induced desorption mass spectrometry, Rev, Phys. Appl., 151 (1980) 1631. 16 D.J. Ashworth, W.M. Baird and C.-J. Chang, J.D. Ciupek, K.L. Busch and R.G. Cooks, Chemical modification of nucleic acids. Methylation of calf thymus DNA investigated by mass spectrometry and liquid chromatography, Biomed. Mass. Spectrom., (1986) in press. 17 M.A. Baldwin and F.W. McLafferty, Direct chemical ionization of relativelyinovolatile samples. Application to underivatized oligopeptides, Org. Mass Spectrom., 7 (1973) 1353. 18 E.L. Esmans, E.J. Freyne, J.H. Vanbroeckhoven and F.C. Alderweireldt, Chemlcal ionization desorption mass spectrometry as an additional tool for the structure elucidation of nucleosides, Biomed. Mass Spectrom., 7 (1980) 377. 19 P.D. Lawley and P. Brookes, Further studies on the alkylation of nucleic acids and their constituent nucleotides, Biochem. J., 89 (1963) 127.